Analysis of Brain Phosphoproteome Using Titanium Dioxide Enrichment and High-Resolution LC-MS/MS

  • Jeffrey M. Sifford
  • Haiyan Tan
  • Hong Wang
  • Junmin PengEmail author
Part of the Neuromethods book series (NM, volume 127)


This chapter outlines the process of performing phosphoproteomic studies of brain tissue: brain dissection, protein extraction and digestion, phosphopeptide enrichment, and peptide identification and quantification by LC-MS/MS. We describe a refined method for rapid, simple, and efficient TiO2-based phosphopeptide enrichment that relies on specific binding of the peptidyl phosphate group and TiO2, with free phosphate competitor added to reduce nonspecific binding. Integration of such a robust phosphopeptide enrichment method, powerful high-resolution LC-MS/MS, and multiplex isobaric labeling enables deep profiling of phosphoproteome with high sensitivity from biological samples, such as the human brain.

Key words

Mass spectrometry LC-MS/MS Proteomics Proteome Phosphoproteome Brain phosphopeptide enrichment Titanium dioxide Isobaric labeling 



The authors thank Drs. Ping-Chung Chen and Yun Jiao for helpful discussion. This work was partially supported by National Institutes of Health grants R01GM114260, R01AG047928, R01AG053987, and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in the St. Jude Children’s Research Hospital Proteomics Facility, partially supported by NIH Cancer Center Support Grant (P30CA021765).


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jeffrey M. Sifford
    • 1
    • 2
  • Haiyan Tan
    • 3
  • Hong Wang
    • 1
    • 2
  • Junmin Peng
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Structural BiologySt. Jude Children’s Research HospitalMemphisUSA
  2. 2.Department of Neurodevelopmental BiologySt. Jude Children’s Research HospitalMemphisUSA
  3. 3.St. Jude Proteomics FacilitySt. Jude Children’s Research HospitalMemphisUSA

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